The present invention relates to cell-free hepadnavirus-derived core particles comprising hepadnavirus core proteins, hepadnavirus polymerase and a nucleic acid such as RNA that is a template for initiation, initial chain elongation or other steps in the replication of a nucleic acid strand encoding at least a portion of the viral genome.
Hepadnaviruses such as hepatitis B virus (xe2x80x9cHBVxe2x80x9d) replicate by a unique pathway that has proved difficult to reconstitute in vitro. The viral genome is a partially duplex DNA. Covalently attached to the 5xe2x80x2 end of the (xe2x88x92) strand is a copy of the viral-encoded polymerase enzyme (P) involved in viral replication. The (xe2x88x92) strand contains the entire viral genome. The ends of the (xe2x88x92) strand are not ligated, but instead are held in proximity to one another by an overlapping (+) strand which, depending on the viral isolate, comprises about 20% to about 80% of the length of the (xe2x88x92) strand. The (+) strand has a short, capped segment of RNA covalently attached at the 5xe2x80x2 end. After infection, the viral genome is believed to migrate to the nucleus of the infected cell, where cellular DNA repair processes are believed to convert it to a closed, circular form, termed xe2x80x9ccccDNAxe2x80x9d. The closed, circular form is then transcribed to create messenger RNA""s encoding viral proteins, including the hepadnavirus polymerase and the hepadnavirus core protein (C) that forms the core particle (i.e., capsid) that encapsidates the viral genetic information (thereby becoming a nucleocapsid). Some of the RNA transcripts are full-length and serve as the template for the replication of the viral genome; these RNA transcripts are known as pregenomic RNA (pgRNA). Hepadnavirus polymerases function (a) as reverse transcriptases, synthesizing the (xe2x88x92) strand of the genomic DNA using the pgRNA as a template, (b) as DNA polymerases, synthesizing the (+) strand of genomic DNA, and (c) as RNase Hs, sequentially digesting portions of the pgRNA template immediately after they have been reverse transcribed. See, Ganem et al. Infectious Agents and Disease 3: 85093, 1994, for a review of the literature on hepadnavirus replication.
The pgRNA typically has the following properties: (a) it is capped; (b) it is greater than genome length; (c) each end (both the 5xe2x80x2 and the 3xe2x80x2 end) has a repeat that includes (i) a sequence element termed xe2x80x9cDR1xe2x80x9d whose 3xe2x80x2 copy is the apparent replication origin of the virus and (ii) a stem-loop-bulge sequence element, termed xe2x80x9cxcex5xe2x80x9d, that contains the true replication origin; and (d) just to the 5xe2x80x2 side of the 3xe2x80x2 repeat there is another copy of the replication origin sequence designated the xe2x80x9cDR2xe2x80x9d element. According to current understanding, in the cytoplasm the hepadnavirus polymerase and pgRNA are encapsidated into the core particle formed from multiple copies of C. Hepadnavirus polymerase interacts with the 5xe2x80x2xcex5 element and reverse transcribes a 3 to 4 base oligomer from the template provided by a sequence within the bulge of the stem-loop-bulge sequence. The first covalent bond in this reverse transcription is formed between a tyrosine hydroxyl of hepadnavirus polymerase and the 5xe2x80x2 phosphate of a deoxynucleotide specified as complementary to a nucleotide of the stem-loop bulge template (see step 1 illustrated in FIG. 1). Thus, in a sense, hepadnavirus polymerase is the xe2x80x9cprimerxe2x80x9d for the initial reverse transcript. This initial bond formation and the subsequent formation of the initial three to four-mer is termed the xe2x80x9cprimingxe2x80x9d reaction. The protein and covalently attached oligomer then migrate to a complementary sequence found in the 3xe2x80x2 DR1 element. This migration step is termed the xe2x80x9ctranslocationxe2x80x9d reaction. From the three or four-mer now base-paired at the 3xe2x80x2 DR1, the polymerase reverse transcribes through to the 5xe2x80x2 end of the pgRNA (see step 2 illustrated in FIG. 1), thereby synthesizing the (xe2x88x92) strand of the viral genome. This reverse transcription step is here termed the xe2x80x9c(xe2x88x92) strand elongationxe2x80x9d reaction. Concurrently with catalyzing the reverse transcription, a separate domain of hepadnavirus polymerase exhibits a RNase H activity that digests the RNA after it has been reverse transcribed into DNA (see step 3 illustrated in FIG. 1). Upon completion of the synthesis of the (xe2x88x92) strand, a 17 to 18 base residue of the 5xe2x80x2 end of the pgRNA including the DR1 sequence remains (see step 4 illustrated in FIG. 1). This residue is translocated to the complementary DR2 element of the (xe2x88x92) strand, and then serves as the primer for the synthesis, again mediated by hepadnavirus polymerase, of a (+) strand priming fragment of the (+) strand complementary to the 5xe2x80x2 end of the (xe2x88x92) strand (see step 5 illustrated in FIG. 1). A portion of this (+) strand priming fragment is also complementary to the 3xe2x80x2 end of the (xe2x88x92) strand and, through this complementarity, the (+) strand priming fragment is used to create a non-covalent bridge linking the two ends of the (xe2x88x92) strand (see step 6 illustrated in FIG. 1). Once the bridge is formed, further (+) strand synthesis proceeds.
In infected cells, hepadnavirus replication occurs inside the viral nucleocapsid. Moreover, genetic studies have implicated C as critical to the process of viral replication in vivo. Nassal, J. Virol. 66: 4107-4116, 1992; Schlicht et al., J. Virol. 63: 2995-3000, 1989; Yu and Summers, J. Virol. 65: 2511-2517, 1991. Nonetheless, it has proved possible, after substantial initial difficulty, to measure some initial replicative activity in vitroxe2x80x94outside of the core particlesxe2x80x94using copies of hepadnavirus polymerase produced by a variety of molecular biology-based techniques. See, for example, Seifer and Standring, J. Virol. 67: 4513-4520; Tavis and Ganem, Proc. Natl. Acad. Sci. USA 90: 4107-4111, 1993; Lanford, J. Virol. 69: 4431-4439, 1995; Seeger, U.S. Pat. No. 5,334,525. However, given the importance of C and core particles to replication in vivo, it is clear that such systems do not faithfully reflect the authentic replication environment and are thus of only limited value as tools for identifying antiviral agents that disrupt viral replication. Furthermore, it is believed that these systems have only a limited capability to elongate minus-strand DNA chains, and that these systems at least in vitro have not been shown to elongate de novo chains of more than, for example, 200 nucleotides.
Others have transfected mammalian cells in xe2x80x9ctransxe2x80x9d, meaning two separate expression vectors were used to express C and polymerase and thus create core particles. The cells were transfected with (a) an expression vector specifying a pgRNA which encodes hepadnavirus polymerase but has a frame-shift mutation making it deficient for the production of C and (b) an expression vector encoding C. See, for example, Bartenschlager et al., J. Virol. 64: 5324-5332, 1990 and Hirsch et al., Nature, 344: 552-555, 1990, both of which articles report mutational studies indicating that hepadnavirus polymerase is needed to correctly package the pgRNA into viral core particles. What this prior work has not done is isolate core particles that are xe2x80x9cfrozenxe2x80x9d in an early stage of the replication process such that the core particles can be used in an in vitro assay that reproduces the intra-core particle environment in which the replication process occurs in vivo. The core particles of this prior work are also believed to have replicated more extensively and have completed much of there (xe2x88x92)-strand synthesis. These prior art core particles thus have reduced reverse transcriptase activity in vitro relative to core particles frozen in a early stage of replication. I
What is needed for determining whether a test compound inhibits early genomic replication mediated by hepadnavirus polymerase is an in vitro system wherein hepadnavirus polymerase operates within the core particle, its natural operative environment, and wherein hepadnavirus polymerase operates from an early stage of the synthesis of genomic DNA through a substantial amount of chain elongation. This need is met by the present invention which provides large quantities of substantially pure viral core particles containing an active hepadnavirus polymerase and a template RNA.
Hepadnavirus capsids have been produced in the recombinant baculovirus/insect cell expression systems. See for example Hildith et al., J. Gen. Virol. 71: 2755-2759, 1990 and Lanford and Notvall, Virology 176: 222-233, 1990. Hepadnavirus capsids have also been produced from recombinant bacteria. See for example Birnbaum and Nassal, J. Virol. 64: 3319-3330, 1990. These systems, however, did not produce core particles containing all three functional components: (1) P; (2) C; and (3) a nucleic acid that serves as a template for useful measurements of an activity of hepadnavirus polymerase. The present invention further provides hapadnaviral core particles with these components, such as recombinant, insect-cell-derived core particles, core particles produced in cells contacted with a reverse transcriptase inhibitor, and core particles that are especially suitable for assays for (+)-strand synthesis.
In one embodiment, the core particles of the compositions of the invention are xe2x80x9cfrozenxe2x80x9d in an early stage of the replication process that produces genomic DNA from pgRNA. This xe2x80x9cfrozenxe2x80x9d state is indicated by several functional measurements, including:
(i) The isolated core particles incorporate added deoxynucleotides into long reverse transcripts (which are preferably DNA molecules linked to the hepadnavirus polymerase), such as reverse transcripts in excess of 400, 2,400 or even 3,000 nucleotides in length. (The longer reverse transcripts are dependent on the presence of a sufficiently long template RNA.)
(ii) Where the template RNA has an xcex5 element and a DR1 element whose first three or four nucleotides (5xe2x80x2 to 3xe2x80x2) are the same as a stretch of three or four nucleotides in the bulge of the xcex5 element, the isolated core particles incorporate added deoxynucleoside triphosphates into long reverse transcripts with apparent origins that align with these three or four nucleotides of the DR1 element.
(iii) The isolated core particles are competent to conduct an authentic priming reaction, which can be measured by measuring the selective incorporation of individual deoxynucleotides into short reverse transcripts in the manner predicted by the sequences of the correct initial, or priming, reverse transcripts. For instance, if the correct priming transcript has the sequence GAA, then when dGTP is added guanosine is selectively incorporated into a conjugate with hepadnavirus polymerase; when dATP is subsequently added it is selectively incorporated into a larger conjugate with hepadnavirus polymerase. Alternatively, the use of the priming template of a template RNA can be confirmed by a comparative mutational analysis. The bulge region of the xcex5 element that defines the priming template can be mutated, which mutation will result in a change in the apparent origin of (xe2x88x92) strand synthesis.
(iv) The isolated core particles are xe2x80x9cfrozenxe2x80x9d in comparison to cores derived via expressing hepadnavirus polymerase and the hepadnavirus core protein in cis (i.e., from a single pgRNA-like mRNA). A useful comparative standard are core particles formed in HepG2.2.15 cells. Such comparative cells appear to have completed HBV (xe2x88x92)-strand DNA synthesis in vivo and in vitro are relatively more active for (+)-strand synthesis than for (xe2x88x92)-strand synthesis. The (+)-strand synthesis is, for example, inhibited by inhibitors of DNA-dependent DNA polymerases such as actinomycin D.
The above functional tests of the frozen state can be conducted in the presence of an effective amount of an inhibitor of DNA polymerase activity (i.e., an activity yielding DNA synthesis from a DNA template), such as in the presence of Actinomycin D, thereby confirming that the activity is reverse transcriptase activity or DNA-dependent DNA polymerase activity. The isolated core particles frozen in an early stage of replication provide tools for assaying whether a biological agent affects (xe2x88x92)-strand synthesis or RNase H activity associated with (xe2x88x92)-strand synthesis.
In a second embodiment, the core particles need not be frozen in an early stage of replication, and provide tools for assaying whether a biological agent affects (+) strand synthesis.
Thus, the invention is directed to these isolated core particles, methods of making these isolated core particles, and to methods of using these isolated core particles to discover or further characterize antiviral agents, as set forth further below.
Preferably, in one embodiment, at least about 0.1% of the isolated core particles of the invention are capable of at least one of the reverse transcriptase activities recited in the paragraph immediately above, more preferably at least about 1%, still more preferably at least about 10%.
The core particle composition of the invention can be prepared with at least about 10-fold more cell-free reverse transcriptase activity, measured for example by the incorporation of labeled deoxynucleoside triphosphates into polymerase conjugates, than core particles isolated from HepG2.2.15 cells. The HepG2.2.15 cell line is among the most widely used constitutively HBV-producing cell lines available for hepadnavirus research. Preferably, the core particles of the invention are at least about 20-fold more active than HepG2.2.15 core particles, more preferably at least about 100-fold.
In a preferred embodiment of the invention, the core particles have at least about 6% of the RNA content of capsids obtained from recombinant bacteria according to the method of Zheng et al., J. Biol. Chem. 267: 9422-9429, 1992 (which document is incorporated herein by reference in its entirety), more preferably at least about 25% of the reference content, and still more preferably at least about 50%.
The invention provides a non-infectious, recombinant hepadnavirus core particle composition comprising isolated hepadnavirus core particles, template RNA encapsidated in the same core particles and hepadnavirus polymerase encapsidated in the same core particles, wherein, upon addition of deoxynucleoside triphosphates to the composition, the hepadnavirus polymerase incorporates deoxynucleotides into reverse transcripts of the template RNA beginning with the first deoxynucleotide of the reverse transcript or within about ten deoxynucleotides, preferably within about three nucleotides, more preferably within about two nucleotides, still more preferably within about one deoxynucleotide, of the first deoxynucleotide of the reverse transcript. Yet more preferably, the hepadnavirus polymerase incorporates deoxynucleotides into reverse transcripts of the template RNA beginning with the first deoxynucleotide of the reverse transcript. Preferably, the hepadnavirus polymerase of the composition, upon addition of deoxynucleoside triphosphates, incorporates deoxynucleotides into reverse transcripts of at least about 400 deoxynucleotides, more preferably at least about 2,400 deoxynucleotides, still more preferably at least about 3,000 deoxynucleotides.
Preferably, in one embodiment of the hepadnavirus core particle composition, the template RNA molecule does not comprise a sequence encoding both the hepadnavirus polymerase and the hepadnavirus C. In one embodiment, the template RNA molecule does not comprise more than one xcex5 element. In another embodiment, the template RNA molecule encodes both hepadnavirus polymerase and hepadnavirus C.
Preferably, the hepadnavirus core particle composition, upon addition of deoxynucleoside triphosphates, incorporates deoxynucleotides into quantities of (+)strand DNA.
Preferably, the template RNA comprises (a) an RNA with an xcex5 element with a priming template and (b) an acceptor site comprising the same sequence as the priming template, and the composition is such that, upon addition of deoxynucleoside triphosphates to the composition, the hepadnavirus polymerase incorporates deoxynucleotides into continuous reverse transcripts of the template RNA of at least about 400 nucleotides that have 5xe2x80x2 ends that align with the acceptor site sequence.
Preferably, template RNA comprises an RNA with an xcex5 element and the composition is such that two separate portions of the hepadnavirus core particle composition, upon the addition of
(a) for a first portion, the deoxynucleoside triphosphate for the predicted initial deoxynucleotide of priming template transcript, or
(b) for a second portion, the deoxynucleoside triphosphate for the predicted initial deoxynucleotide of the priming template transcript and the deoxynucleoside triphosphate for the predicted second distinct deoxynucleotide utilized in the priming template transcript,
synthesize, respectively, a first adduct with the polymerase and a second, larger adduct with the polymerase, and wherein the quantity of both the first adduct and the second adduct is at least about two-fold greater than the quantity of polymerase adducts formed when one or both of the other two deoxynucleoside triphosphates are used.
For use in the bioactive agent screening aspect of the invention, the composition can comprise a candidate bioactive agent. This aspect of the invention comprises identifying bioactive agents that interrupt or inhibit hepadnavirus replication or characterizing the potency of antiviral agents in interrupting or inhibiting hepadnavirus replication by (a) adding one or more deoxynucleoside triphosphates to a core particle composition that contains a candidate bioactive agent and (b) detecting the formation of reverse transcripts or detecting the size of the reverse transcript. Preferably, the added deoxynucleoside triphosphates are labeled and the detecting step comprises determining the amount of label incorporated into reverse transcripts or determining the size of reverse transcripts having associated label. Preferably, the added deoxynucleoside triphosphates are labeled with a radioisotope, a chromophore or a fluorescent molecule, and the detecting step comprises detecting radioactivity, chromophore-created optical density or fluorescent molecule-created fluorescence incorporated into reverse transcripts. Preferably, the detecting step comprises separating core particles containing reverse transcripts from unincorporated deoxynucleosides by precipitating the core particles. The precipitation can be acid precipitation. The detecting step can comprise contacting the reverse transcripts with a protease to digest away any protein conjugated with the reverse transcripts.
The method can further comprise comparing the amount of reverse transcript to the amount formed when the method is replicated in all aspects except that the candidate bioactive agent is omitted. Preferably, the template RNA in the core particle composition comprises an xcex5 element with a priming template, the method further comprising measuring the priming reaction by adding a subset of deoxynucleoside triphosphates that allows the addition one or more of the priming template-directed nucleotides. In another embodiment, the template RNA in the core particle composition comprises an xcex5 element and a DR1 element, and the method further comprises measuring those continuous reverse transcripts that have 5xe2x80x2 ends that begin with an apparent DR1-contained origin of replication.
The invention further provides a method of preparing a hepadnavirus core particle composition comprising (a) hepadnavirus core particles, (b) template nucleic acid encapsidated in core particles and (c) hepadnavirus polymerase encapsidated in core particles, wherein, upon addition of deoxynucleoside triphosphates to the composition, the hepadnavirus polymerase incorporates deoxynucleotides from the added deoxynucleosides into reverse transcripts of the template nucleic acid beginning with the first deoxynucleotide of the reverse transcript or beginning within about ten deoxynucleotides of the first deoxynucleotide of the reverse transcript, the method comprising (1) transfecting or infecting a cell with one or more nucleic acid vectors that (i) encode hepadnavirus polymerase and express hepadnavirus polymerase in the transfected or infected cell and (ii) encode hepadnavirus C and express hepadnavirus C in the transfected or infected cell, and (2) isolating (for instance by disrupting cells) to release said core particles formed from the expressed hepadnavirus C and hepadnavirus polymerase and the template nucleic acid, which template nucleic acid is derived from one of the nucleic acid vectors. Preferably, (a) the mRNA transcript from the hepadnavirus polymerase encoding sequence or the mRNA transcript from the hepadnavirus C encoding sequence is the template nucleic acid, (b) the template RNA comprises an xcex5 element, and (c) the template RNA is encapsidated in the core particles that are isolated from the cells. In one embodiment, the transfected or infected cell is an insect cell and the vector is a baculovirus vector. In another embodiment, the transfected or infected cell is a mammalian cell and the vector is a mammalian expression vector. In yet another embodiment, the transfected or infected cell is an yeast cell and the vector is a yeast expression vector. In still another embodiment, the transfected or infected cell is an bacterial cell and the vector is an bacterial expression vector.
The method can further comprise separating the core particles from cellular components of differing sizes and densities by centrifugation. The method can comprise separating the core particles from cellular components of differing densities by density gradient centrifugation or, the method can comprise, digesting the cellular components with enzymes such as proteases or nucleases to which the core particles are resistant.
In one embodiment, the method comprises, or the method can comprise, digesting the cellular components with enzymes such as proteases or nucleases to which the core particles are resistant, growing the transfected or infected cell in the presence of a hepadnavirus polymerase-inhibiting effective amount of a reverse transcriptase inhibitor. In one embodiment, the hepadnavirus polymerase-encoding nucleic acid is on a first vector and hepadnavirus C-encoding nucleic acid is on a separate, second vector.
In certain embodiments the invention provides non-infectious, recombinant hepadnavirus core particle composition that is:
(a) isolated from cells transformed with one or more recombinant vectors encoding hepadnavirus core protein and hepadnavirus polymerase and contacted with a reverse-transcriptase inhibiting effective amount of a reverse transcriptase inhibitor; or
(b) isolated from insect cells transformed with one or more recombinant baculoviruses encoding hepadnavirus core protein and hepadnavirus polymerase;
wherein the core particles comprise hepadnavirus polymerase and nucleic acid encapsidated therein such that, upon addition of deoxynucleoside triphosphates to the composition, the deoxynucleotides are incorporated into DNA. Preferably, a first recombinant vector encodes the core protein and a second vector encodes the polymerase. In one embodiment, the encapsulated nucleic acid includes RNA, and the addition of deoxynucleotides results in (xe2x88x92)-strand synthesis. In another embodiment, the addition of deoxynucleotides results in (+)-strand synthesis.
In other embodiments, the invention provides a non-infectious, recombinant hepadnavirus core particle composition comprising core particles that comprise hepadnavirus polymerase and nucleic acid encapsidated therein such that, upon addition of deoxynucleoside triphosphates to the composition, deoxynucleotides are incorporated into a substantial distribution of (+)-strand nucleic acids. A preparation of (+)-strand nucleic acids has a xe2x80x9csubstantialxe2x80x9d distribution of nucleic acids if for example at least about 0.5% have size from about 0.1 to about 1.0 kb, preferably for about 0.1 to about 3.0 kb. More preferably, at least about 5% of the nucleic acids have molecular weights between about 0.1 and about 1.0 kb, and yet more preferably between 0.1 and about 3.0 kb. Still more preferably, at least about 50% of the nucleic acids have molecular weights between about 0.1 and about 1.0 kb, and yet more preferably between 0.1 and about 3.0 kb.
The core particles of any embodiment of the invention can be used in a method of identifying bioactive agents that interrupt or inhibit hepadnavirus replication or characterizing the potency of antiviral agents in interrupting or inhibiting hepadnavirus replication, the method comprising
(1) adding one or more deoxynucleoside triphosphates to the core particle composition;
(2) adding a bioactive agent to the core particle composition; and
(3) following steps (1) and (2), either (i) detecting formation of nucleic acids or detecting sizes of nucleic acids found in the core particle composition or (ii) measuring an RNase H activity exhibited by the core particle composition.
The invention is described with reference to three primary aspects: the core particle composition, the bioactive agent screening method and the production method. These are closely intertwined and it will be recognized that all preferred or alternate embodiments of the composition can be used in the screening method or produced by the production method, or that any preferred core particle recited with respect to the screening method or the production method is a part of the core particle composition aspect of the invention.